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10.1039/C5GC00712G.

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DOI: 10.1039/C5GC00712G
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Green Chemistry Accepted Manuscript

ARTICLE

The magic of aqueous solutions of ionic liquids: Ionic liquids as a

powerful class of catanionic hydrotropes
Received 00th January 20xx, Ana Filipa M. Cláudioa, Márcia C. Nevesa, Karina Shimizub,c, José N. Canongia Lopesb,c, Mara G. Freirea
Published on 21 May 2015. Downloaded by North Dakota State University on 22/05/2015 04:39:47.

Accepted 00th January 20xx and João A. P. Coutinhoa*

DOI: 10.1039/x0xx00000x
Hydrotropes are compounds able to enhance the solubility of hydrophobic substances in aqueous media and
www.rsc.org/ therefore are widely used in the formulation of drugs, cleaning and personal care products. In this work, it is shown
that ionic liquids are a new class of powerful catanionic hydrotropes where both the cation and the anion
synergistically contribute to increase the solubility of biomolecules in water. The effects of the ionic liquid chemical
structures, their concentration and the temperature on the solubility of two model biomolecules, vanillin and gallic
acid, were evaluated and compared with the performance of conventional hydrotropes. The solubility of these two
biomolecules was studied in the whole composition range, from pure water to pure ionic liquids, and an increase
in the solubility of up to 40-fold was observed, con-firming the potential of ionic liquids to act as hydrotropes. Using
Dynamic Light Scattering, NMR and molecular dynamics simulations, it was possible to infer that the enhanced
solubility of the biomolecule in the IL aqueous solutions is related to the formation of ionic-liquid-biomolecules
aggregates Finally, it was demonstrated that hydrotropy induced by ionic liquids can be used to recover solutes
from aqueous media by precipitation, simply by using water as anti-solvent. The results here reported have a
significant impact in the understanding of the role of ionic liquids aqueous solutions in the extraction of added-
value compounds from biomass as well as in the design of novel processes for their recovery from aqueous media.

than in traditional surfactants and they do not form micelles

Introduction
Advances in the dissolution of poorly soluble compounds in
aqueous media play an important role in the formulation of
drugs, cleaning agents and personal care products.1-4 Moreover,
they are also of pivotal relevance in related industrial processes,
such as crystallization and purification.5 Hydrotropes are
compounds used to increase the concentration levels of
hydrophobic solutes in aqueous solutions. Their ability to
dramatically increase the solubility of sparingly soluble organic
compounds in water was already demonstrated. 6,7 They are a
class of highly water soluble salts or molecules characterized by
an amphiphilic structure. Conventional hydrotropes used by
industry are typically composed of a phenyl (hydrophobic part)
attached to an anionic group (hydrophilic part) where
ammonium, calcium, potassium or sodium act as counter ions. 4
Although hydrotropes are used to improve the solubility of
poorly soluble compounds in water they are not surfactants; the
apolar part, or hydrophobic moiety, of hydrotropes is smaller
a. CICECO - Aveiro Institute of Materials, Chemistry Department, University of
Aveiro, 3810-193 Aveiro, Portugal
b. Centro de Química Estrutural, Instituto Superior Técnico, 1049 001 Lisboa,
Portugal
c. Instituto de Tecnologia Química e Biológica, UNL, AV. República Ap. 127, 2780
neither do they present a critical micellar concentration (CMC)
in absence of a solute.8,9 In addition to the increase on the
solubility, hydrotropes may also play a role in the stabilization
of aqueous solutions, on the tailoring of their viscosity, and by
modifying the liquid-liquid phase separation temperatures. 5
The hydrotropy phenomenon was first reported by Neuberg in
1916.7 The author demonstrated the increased solubility of
sparingly soluble compounds in water by the addition of alkali
metal salts of various organic acids with short alkyl chains.
Albeit a large body of work during the past century addressed
this phenomenon, its mechanism of action is still not fully
understood.7 In the past decades, three main theories to justify
the increased solubility of target compounds in water by the
role of hydrotropes were proposed.2 Some authors11-13
suggested that the hydrotropic-mediated solubilisation results
from changes in the nature and structure of the solvent, i.e.,
that a disruption of the water structure induced by the
hydrotropes occurs, allowing an increase in the solubility of the
molecule of interest; The formation of solute-hydrotropes
complexes has also been proposed to explain the enhanced
solubility.14-17 According to this theory, the increase of solubility
is the result of a collective molecular phenomenon that takes
place by the formation of molecular complexes between the
hydrotrope and the solute; 14-17 More recent studies18-24

901 Oeiras, Portugal

proposed that the co-aggregation of the solute with the
† Electronic Supplementary Information (ESI) available: supporting figures and hydrotropes is the main mechanism behind the enhanced
tables were reported in supporting Information. See DOI: 10.1039/x0xx00000x solubilisation observed.

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 1

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Hydrotropes are interesting compounds from a green chemistry as sodium benzoate, sodium citrate and sodium View thiocyanate.
Article Online
perspective as they can replace hazardous co-solvents and The molecular-level mechanism ruling DOI:the
10.1039/C5GC00712G
hydrotropicity
present, in general, a low toxicity and have a low induced by ionic liquids is also proposed and discussed based on

Green Chemistry Accepted Manuscript

bioaccumulation potential - their octanol-water partition experimental evidence obtained from Dynamic Light Scattering
coefficients, due to their hydrophilic nature, are usually lower (DLS) and NMR experiments and molecular dynamics
than 1.0.25 Moreover, and since the solubility of a solute in simulations.
aqueous media depends on the concentration of the
hydrotrope, their recovery can be easily achieved using water
as the anti-solvent (the greenest of all solvents). In this context,
the purity of the final product can be greatly improved by
further washing with water.26
The good solvation ability of ionic liquids (ILs) for a wide range
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of solutes is well-known, and therefore, they have been

proposed as potential substitutes for the conventional organic
solvents employed in extraction and separation processes. 27-29 Fig 1. Chemical structures of (i) vanillin and (ii) gallic acid.
It has been shown that in aqueous solution the nanostructure
of ILs is, to a fair extent, maintained down to concentrations as Finally, the importance and relevance of the results here
low as 0.1 mole fraction, and that IL-based aqueous biphasic obtained are discussed in terms of their applicability in
systems display a tailored and remarkable extraction extraction and purification processes of biomolecules from
performance for a wide variety of compounds when compared biomass sources, where the design of novel cost-effective and
with conventional polymer-based systems.30 Furthermore, environmentally friendly processes could be envisaged.
while aqueous solutions of ILs have shown to be good solvents
for the extraction of added-value compounds from biomass,31
the use of ILs as hydrotropes has never, to the best of our Experimental Section
knowledge, been previously described or investigated. Chemicals
Previous works on the use of ILs for the extraction and
A large variety of ionic liquids (ILs) was studied in this work to
purification of phenolic compounds, such as gallic acid and
cover the effects of the cation and anion type and the cation
vanillin, have been reported.28,29,32-34 The large interest in
chain length. The ILs investigated are 1-ethyl-3-
vanillin results from its relevant properties for the human
methylimidazolium chloride, [C2C1im]Cl, 1-ethyl-3-
health, namely due to its antioxidant, anti-inflammatory, radical
methylimidazolium dicyanimide, [C2C1im][N(CN)2], 1-butyl-3-
scavenger and antimicrobial characteristics.29 Likewise, gallic
methylimidazolium trifluoromethanesulfonate,
acid is a phenolic compound with important properties in the
[C4C1im][CF3SO3], 1-butyl-3-methylimidazolium thiocyanate,
health and nutrition fields – a result of its anti-inflammatory,
[C4C1im][SCN], 1-butyl-3-methylimidazolium methylsulfate,
antibacterial, antifungal, antioxidative, phytotoxic and radical
[C4C1im][CH3SO4], 1-butyl-3-methylimidazolium tosylate,
scavenging activities.28,35 These biomolecules are present in
[C4C1im][TOS], 1-butyl-3-methylimidazolium bromide,
high concentrations in a variety of biomass sources, such as
[C4C1im]Br, 1-butyl-3-methylimidazolium dicyanamide,
fruits, vegetables and wood, and also in many residues from
[C4C1im][N(CN)2], 1-butyl-3-methylimidazolium chloride,
industrial or agricultural activities.25,36-38 Their extraction from
[C4C1im]Cl, 1-hexyl-3-methylimidazolium chloride, [C6C1im]Cl,
natural sources is usually carried out with mildly polar solvents
1-methyl-3-octylimidazolium chloride, [C8C1im]Cl, 1-decyl-3-
such as alcohols39,40, due to their low solubility in water (the
decylimidazolium chloride, [C10C1im]Cl, 1-dodecyl-3-
solubility of vanillin in water is 12.78 g.L-1 and of gallic acid is
methylimidazolium chloride, [C12C1im]Cl, 1-tetradecyl-3-
12.14 g.L-1 at room temperature).41 Although no previous
methylimidazolium chloride, [C14C1im]Cl, 1-butyl-3-
studies on the hydrotropic solubilization of gallic acid were
methylpyridinium dicyanamide, [C 4C1py][N(CN)2], 1-butyl-1-
reported, the solubility of vanillin in aqueous solutions is
methylpiperidinium chloride, [C4C1py]Cl, 1-butyl-1-
already recognized to increase in the presence of typical
methylpiperidinium chloride, [C4C1pip]Cl, 1-butyl-1-
hydrotropes, such as nicotinamide, sodium salicylate, resorcinol
methylpyrrolidinium chloride, [C4C1pyrr]Cl,
and citric acid.42
tetrabutylammonium chloride, [N4444]Cl, tetrabutyl-
With the amphiphilic behaviour of ILs in mind,43 that seems to
phosphonium chloride, [P4444]Cl, tributylmethylphosphonium
be the driving force behind their ability to dissolve a wide range
tosylate, [P4441][TOS], cholinium chloride, [N1112(OH)]Cl, and
of compounds, the potential of ILs to act as hydrotropes was
tetrabutylammonium tosylate, [N4444][TOS]. The imidazolium-,
here investigated based on their ability to enhance the solubility
pyridinium-, and pyrrolidinium-based ILs were purchased from
in water of two phenolic compounds, namely vanillin and gallic
Iolitec. The tetrabutylphosphonium chloride and
acid, whose structures are depicted in Figure 1. The hydrotropic
tributylmethylphosphonium tosylate were kindly offered by
behaviour of ionic liquids was here evaluated by comparing the
Cytec Industries Inc. Tetrabutylammonium chloride,
solubility of the two antioxidants in aqueous solutions of ILs
tetrabutylammonium tosylate and cholinium chloride were
with that in pure water, pure ILs, and aqueous solutions of
obtained from Sigma-Aldrich. All ILs used have a stated supplier
common hydrotropes and some salting-in inducing salts, such

2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

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DOI: 10.1039/C5GC00712G
Journal Name

Green Chemistry Accepted Manuscript

ARTICLE

Cations
1-alkyl-3- N,N- dialkyl N,3-dialkyl
alkylimidazolium pyrrolidinium N+
pyridinium
[CnCnim] + [CnCnpyrr] + R2 R1
[Cn-3-Cnpy] +
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R1 R1
N,N- dialkyl tetraalkyl tetralkyl
piperidinium R4 P+ R2 phosphonium R4 N+ R2 ammonium
[CnCnpip] + [Pnnnn] + [Nnnnn] +
R3
R3

Choline
HO N+
[N1112OH]+ Na+ sodium

Anions
F
- triflate
Sthiocyante
Cl- chloride Br- bromide N - [SCN]
F F
(trifluoro
O S O methanesulfonate)
O- [CF3SO3] -
O
O
Tosylate N
Dicyanamide Methylsulfate
O S [TOS]- -
O S O
[N(CN)2]- [CH3SO4] -
O N N
O

Citrate
Benzoate [C6H5O7]3-
[C7H5O2]-

Fig. 2 Chemical structures of the anions and cations of all ILs and salts investigated.

purity of at least 98 wt%. Before use, aiming at reducing the

water and volatile compounds contents to negligible values, all
IL samples were dried under constant agitation and vacuum
(10-2 Pa) at a temperature of 323 K for a minimum of 48 h. After
this procedure, the purity of each IL was further checked by 1H
and 13C NMR and confirmed to be > 98 wt %. In ESI is reported
a summary table of the chemicals used in this work (Table S1).
The deuterium oxide used was acquired from Aldrich with >
99.96 % D atoms. The 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid
sodium salt (TSP) was from Aldrich with > 98 % D atoms. Sodium
benzoate (NaC7H5O2), > 99.0 wt % pure, was supplied by
Panreac, sodium thiocyanate (NaSCN), > 98.0 wt % pure, was
supplied by Fluka, sodium chloride (NaCl) > 98.0 wt % pure, was
from ChemLab, sodium citrate (Na3C6H5O7), > 98.0 wt % pure,
was from JMGS, sodium tosylate Na[TOS] > 95.0 wt % pure, was
from TCI and sodium dicyanamide Na[N(CN)2], > 96.0 wt % pure,
was from Sigma-Aldrich. All of these compounds were also dried
before use. The chemical structures of anions and cations of all
ILs and salts investigated are depicted in Figure 2.
Vanillin, > 99 wt % pure, was supplied by Acros and gallic acid, >
99.5 wt% pure, was acquired from Merck. Both antioxidants
were used as received.
The water employed was double distilled, passed across a
reverse osmosis system, and further treated with a Milli-Q plus
185 water purification apparatus.
Solubility of antioxidants
Each antioxidant (vanillin and gallic acid) was added in excess
amount to each IL aqueous solution, pure water or pure IL, and
was then equilibrated on an air oven (at given temperatures (±
0.5 K)) under constant agitation using an Eppendorf
Thermomixer Comfort equipment. Previously optimized
equilibration conditions were established: stirring velocity of
750 rpm and equilibration time of at least 72 h. After the
saturation was reached, all samples were centrifuged at the
same temperature of equilibration in a Hettich Mikro 120
centrifuge during 20 minutes at 4500 rpm to separate the
macroscopic solid and liquid phases. After centrifugation, all
samples were put in an air bath equipped with a Pt 100 probe
and PID controller at the temperature used in equilibrium
assays during 2 h. Then, the samples of the liquid phase were
carefully collected and diluted in ultra-pure water, and the
amount of vanillin and gallic acid was quantified through UV-
spectroscopy, using a SHIMADZU UV-1700, Pharma-Spec
Spectrometer, at a wavelength of 280 nm and 262 nm,
respectively, using calibration curves previously established. In
all experiments, control samples at the same compositions, but
without antioxidant, were used. At least three individual
samples were quantified for each mixture and temperature.

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However, in some systems, the mixture composed of
antioxidant, IL and water created a biphasic liquid-liquid
system. In these situations, the amount of IL and vanillin in both
the top and bottom phases was quantified by UV-spectroscopy
as described before.
Dynamic light scattering (DLS)
To evaluate the presence of IL-solute aggregates, as well as to
determine their size, solutions composed of [C 4C1im][TOS],
water and vanillin were analysed by dynamic light scattering
(DLS), using a Malvern Zetasizer Nano-ZS from Malvern
Instruments. Samples were irradiated with red light (HeNe
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laser, wavelength of 565 nm) and the intensity fluctuations of
the scattering light (detected at a backscattering angle of 173°)
were analysed to obtain an autocorrelation function. The
respective software (DTS v 7.03) provides the particles’ size
average and their distribution. The radii of each aggregate was
determined from the DLS measurements using the Stokes–
Einstein equation assuming spherical aggregates, and a low
volume fraction of the dispersed phase. Consequently, the
determined values must be considered with caution and
regarded as approximate ones. Samples were measured in
disposable polystyrene cuvettes at a temperature of 298 K. Data
were then acquired in the automatic mode, ensuring that
enough photons were accumulated for the result to be
statistically relevant. The software also incorporates a ‘data
quality report’. The solution viscosities and refractive indexes
were previously measured to the DLS measurements.
Two types of experiments were carried out. In the first set,
aqueous solutions of 0.80 mol.kg-1 (IL/water) of [C4C1im][TOS]
were prepared. Then, this stock solution was used to prepare
different solutions with variable amounts of vanillin, namely
0.73, 0.62, 0.55, 0.49, 0.44, 0.40, 0.36 and 0.34 mol.kg-1. In the
second set, an aqueous solution containing 0.64 mol.kg-1 of
[C4C1im][TOS] and 0.73 mol.kg-1 of vanillin was diluted with
pure water aiming at maintaining the hydrotrope and vanillin at
a constant ratio. In order to avoid the presence of micrometric
aggregates and to remove all the dust particles, before the
measurements, all solutions were homogenized in an ultrasonic
bath and then filtered using a 0.2 mm PTFE membrane.
Nuclear magnetic resonance (NMR) spectroscopy
The 1H NMR spectra were obtained for several samples placed
in NMR spectroscopy tubes containing sealed reference
capillaries with D2O and TSP as the internal reference, and at
298 K. The 1H NMR measurements were performed on a Bruker
Avance 300 spectrometer operating at 300.13 MHz.
Molecular Dynamics Simulation (MD)
Two ionic liquids, [C4C1im][N(CN)2] and [C4C1im][SCN], were
tested using Molecular Dynamics (MD) simulations. They were
parameterized using the CL&P atomistic force field44-46, which is
an extension of the AMBER and OPLS force fields47 specially
designed to study ionic liquids and their homologous series.
The water and vanillin molecules were modeled using the SPC
model48 and the OPLS force field, respectively. Molecular
4 | J. Name., 2012, 00, 1-3
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dynamics simulations were carried out using the DL_POLY 2.20
View Article Online
package. 49he runs were performed with DOI:cubic10.1039/C5GC00712G
boxes with sides
of at least 5 nm, 2 fs time-steps and 2 nm cutoff distances. Ewald
Green Chemistry Accepted Manuscript
summation corrections were performed beyond those cutoffs.
The IL aqueous solutions were modeled using either 101
[C4C1im][N(CN)2] ion pairs or 105 [C4C1im][SCN] ion pairs mixed
with 4600 water molecules. The vanillin-water mixture was
modeled using 100 vanillin molecules and 4600 water
molecules. Finally, the vanillin-IL-water systems were modeled
using the aforementioned quantities of IL and vanillin mixed
with 4600 water molecules.
All simulations started from low-density configurations that
were subjected to equilibration runs under isobaric isothermal
ensemble conditions (p= 0.1 MPa and T= 373 K with
Nosé−Hoover thermostats and barostats with relaxation time
constants of 1 and 4 ps, respectively). After 1.3 ns, the density
of each system reached constant and consistent values,
indicating that equilibrium had been attained and possible
ergodicity problems had been overcome. Finally, several (at
least six) consecutive production stages of 1.0 ns each were
performed, and the combined results were used for the
interaction, structural and aggregation analyses of the studied
systems.
Interaction and Structural Analyses
The pair radial distribution functions gij(r) between selected
pairs of atoms or interaction centers were calculated up to r =
2.5 nm distances in the usual way.50 Total static structure
factors, S(q), were also calculated from the MD trajectory data
using a previously described methodology. 51
Aggregation Analyses
The aggregation analyses of the [C4C1im][N(CN)2], [C4C1im][SCN]
ILs and their mixtures with water and vanillin focused on five
types of issues: (i) the evaluation of the connectivity between
the charged moieties of the molecular ions that compose the
so-called polar network; (ii) the estimation of the interactions
between the IL ions and the water molecules; (iii) the
calculation of the aggregation state between the IL alkyl side
chains; (iv) the evaluation of the connectivity among the vanillin
molecules and an estimation of their aggregate size; and (iv) the
calculation of the connectivity between the IL ions and the
vanillin molecules.
All these types of connectivity analysis are based on previously
described algorithms52,53 that generate neighbor lists for
selected interaction centers, in a three-stage sequential
process:First, the different types of interaction centers are
defined. In the analyses of type i) the selected interaction
centers are the center of mass of the imidazolium ring of the
cation (im) and the center of mass of anion. For type ii) analyses
we have selected the oxygen atom of the water molecule and
all nitrogen atoms of the [N(CN]2] anion or the nitrogen and
sulfur atoms of the [SCN] anion. Type iii) analyses were
conducted considering all the carbon atoms of the butyl chain
of the cation, except the carbon atom directly connected to the
imidazolium ring. For type iv) analyses the selected interaction
centres are all non-hydrogen atoms in the vanillin molecules.
Finally, type v) analyses were conducted by selecting all non-
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hydrogen atoms in the cation or anion of the IL and all non-
hydrogen atoms in the vanillin molecule;
Second, a connectivity threshold for each case is established by
considering the corresponding gij(r) data between the selected
interaction centres.52 In case i) such threshold was set to 0.8 nm,
corresponding to the first coordination shell limit of the gij(r)
data between the center of mass of the imidazolium ring of the
cation (im) and the center of mass of anion. In case ii) the
threshold was set to 0.35 nm (based on the water-anion
correlation data), and in cases iii) to v) the threshold was set to
0.5 nm, corresponding to average intermolecular contact
distances between non-hydrogen atoms evaluated from the
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corresponding g(r) functions;
Third, the use of the threshold criteria allows the computation
of closest-neighbor lists for each interaction center for all
recorded configurations in the MD trajectories, thus
ascertaining the connectivity between the selected species.
When the interaction centers belong to different species —
cation-anion in case i), anion-water in case ii) and ion-vanillin in
case v) — the analyses will yield aggregates with a built-in
alternation between species (e.g., in a polar network cations are
directly connected to anions and vice-versa). In the case of
interaction centers belonging to species of the same type —
butyl-butyl in case iii) and vanillin-vanillin in case iv) — the
corresponding aggregates correspond to clusters containing a
single type of molecule/residue.
Finally, five different statistical functions were used to
characterize the network/aggregates that emerge from the
connectivity lists.52-53 These include (i) P(na), the discrete
probability distribution function of finding a given interaction
centre in an aggregate of a given size (composed of na species);
(ii) Ni, is the average number of first-contact neighbors of a
interaction centre; (iii) Ni(na), the average number of neighbors
within an aggregate of size na ; (iv) Rd(na), the ratio between the
longest distance between two atoms belonging to the same
aggregate of size na and the simulation box diagonal; and (v)
RV(na), the ratio between the apparent volume of an aggregate
of size na and the volume of the simulation box. These five
statistical tools have been introduced and described in more
detail elsewhere.52
Results and Discussion
Aiming at investigating the potential of ILs to act as
hydrotropes, the solubilities of vanillin and gallic acid were
determined in several aqueous solutions with variable
concentrations of ILs and compared with the results obtained
with common hydrotropes and salting-in inducing salts. The
solubilities of vanillin and gallic acid at 303 K in pure water,
measured in this work, are (11.12 ± 0.03) g.L -1 (0.073 mol.L-1)
and (14.38 ± 0.41) g.L-1 (0.084 mol.L-1), respectively, and are in
good agreement with data previously reported in the
literature.41,54,55 All the detailed data along with the respective
standard deviations are presented in Supporting Information-
Table S2.
Effect of the IL concentration
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COMMUNICATION
The gallic acid solubilities in aqueous solutions of
View Article Online
[C4C1im][N(CN)2] and [C4C1im]Cl, and DOI: 10.1039/C5GC00712G
vanillin in aqueous
solutions of [C2C1im][N(CN)2], [C4C1im][TOS] and [C4C1im]Cl
Green Chemistry Accepted Manuscript
were studied in the whole concentration range, from pure
water up to aqueous saturated solutions of these compounds
at 303 K. The influence of the ILs concentration in the solubility
of gallic acid and vanillin is depicted in Figure 3. The detailed
values of solubility, and respective standard deviations, are
reported in Supporting Information- Table S2. The graphical
representation in other units, namely concentration of
biomolecule (mol.kg-1) vs. concentration of hydrotrope (mol.kg-
1), and concentration of biomolecule (g.kg -1) vs. weight fraction
of hydrotrope (wt%), are also presented in Supporting
Information- Figure S1.
S and S0 represent the solubility (mol.kg-1) of each biomolecule
in the aqueous solutions of hydrotrope and in pure water,
respectively; therefore, S/S0 represents the solubility
enhancement.
50
(S/S0) Biomolecule
40
30
20
10
0
0 1 2 3 4 5 6
[IL] / mol.kg-1
Fig. 3 Influence of the ILs concentration in the solubility of gallic acid in aqueous
solutions of () [C4C1im][N(CN)2], and () [C4C1im]Cl; and vanillin in aqueous
solutions of () [C2C1im][N(CN)2], (▲) [C4C1im][TOS] and (×) [C4C1im]Cl at 303 K.
Lines are guides for the eye.
The results obtained show a synergetic effect of the two
solvents on the solubility of the two antioxidants, with aqueous
solutions of IL displaying a much higher capacity to solubilize the
antioxidants than any of the two pure solvents, and with
solubility enhancements that may reach 40-fold. These results
are thus clear evidence of the exceptional capacity of ILs to act
as hydrotropes. In an attempt to determine the minimum
hydrotrope concentration (MHC), also referred to as the critical
aggregate concentration (cac) by some authors56, for
[C4C1im]Cl, [C4C1im][TOS], and Na[TOS], the solubility of vanillin
was measured in aqueous solutions with salt concentrations
ranging between 0.01 and 1.5 mol.kg-1 (hydrotrope/water). The
results reported in Figure 4 do not allow the clear identification
of a MHC for any of the IL-related hydrotropes investigated,
including Na[TOS]. These results show, in agreement with
various authors,10,56,57 a continuous variation in the solubility
instead of a clear change in behaviour attributable to a critical
phenomenon. While the MHC concept has been extensively
used in the interpretation of the hydrotropic behaviour, it has
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pure water, () [C2C1im]Cl, () [C4C1im]Cl, () [C6C1im]Cl, () [C8C1m]Cl, ()
recently been dismissed by several authors that relate it with [C10C1im]Cl, () [C12C1im]Cl, (▬) [C14C1im]Cl, () [C2C1im][N(CN) 2],Online
View Article ()
[C4C1im][N(CN)2], () [C4C1py][N(CN)2], () [C4C1py]Cl,DOI: () [C4C1pyrr]Cl, ()
10.1039/C5GC00712G
less accurate experimental measurements (as demonstrated by [C4C1pip]Cl, () [C4C1im][TOS], () [C4C1im][SCN], () Na[C 7H5O2], () Na[SCN],
(▬) Na[C6H5O7], () NaCl, () [N4444]Cl, (+) Na[TOS], () [P4444]Cl, ()
Subramanian and Anisimov58-60, showing that “droplets in [N4444][TOS], (+) [P4441][TOS], and () Na[N(CN)2]. Lines are guides for the eye.

Green Chemistry Accepted Manuscript

aqueous solutions of hydrotropes are caused by the
contamination of the non-ionic hydrotrope by trace amounts of For a more detailed analysis of the IL structural features (anion
hydrophobic impurities”) or to a faulty interpretation of the nature, cation core and cation alkyl side chain length), the
experimental data.57,61 Russo and Hoffmann57 also showed that Setschenow equation62 was used:
NMR chemical shift and surface tension data are not applicable log( S )  K  C
S0 Hyd Hyd
(1)
to determine the MHC of short-chained ILs. According to these
authors57, these compounds “form aggregates of less defined where S and S0 are, respectively, the solubility (mol.kg -1) of each
size and shape and in more gradual concentration dependent biomolecule in the hydrotrope aqueous solution and in pure
transitions than is known to occur for surfactant molecules”. water, CHyd is the concentration of hydrotrope in aqueous
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Attempts carried out in this work to establish the MHC of some solution (mol.kg-1) and KHyd is the hydrotropy constant (kg-
1.mol). The hydrotropy constants, K
[CnC1im]Cl, with n ≤ 6, by surface tension measurements Hyd, were estimated for each
confirm the results reported by Russo and Hoffmann57 (cf. system studied and are reported, along with their standard
Supporting Information-Figure S2). deviations, in Table 1.
1.0
1.0
-1

Table 1 KHyd values for the various hydrotropes studied in the solubility of vanillin and
mol.kg-1
[Vanillin]//mol.kg

gallic acid at 303 K ± 0.5 K.

Selectivity =
[Vanillin]

Hydrotrope KHyd (kg-1.mol) ± σ

0.5
0.5 KHyd Van/ KHyd GA

Vanillin Gallic acid

[C2C1im]Cl 0.406 ± 0.006

0.0
0.0
0.01
0.01 0.10
0.10 1.00
1.00 10.00
10.00 [C4C1im]Cl 0.706 ± 0.014 0.786 ± 0.041 0.90
-1 -1
[Hyd]
[hyd] /mol Hyd.kg
/ molhyd .kgwater
water [C6C1im]Cl 0.966 ± 0.031
[C8C1im]Cl 1.344 ± 0.042 0.885 ± 0.013 1.52
Fig.4 Influence of the ILs concentration in the solubility of vanilin in aqueous
solutions of (●) [C4C1im]Cl, () [C4C1im][TOS] and () Na[TOS] to evaluate the [C10C1im]Cl 1.365 ± 0.064
minimum hydrotrope concentration (MHC). Lines are guides for the eye.
[C12C1im]Cl 1.442 ± 0.038
Effect of the IL chemical structure [C14C1im]Cl 1.488 ± 0.023

In the studies reported hereafter, aqueous solutions of ILs with [C4C1im]Br 0.646 ± 0.012
concentrations up to 1.6 mol.kg-1 (hydrotrope/water) were [C4C1im][SCN] 0.978 ± 0.017* 0.703 ± 0.016 1.39
used to study the impact of the IL structural features upon its [C4C1im][TOS] 1.584 ± 0.053 1.035 ± 0.048 1.53
ability to enhance the solubility of the investigated antioxidants. [C4C1im][CH3SO4] 0.808 ± 0.031
The results for a range of ILs and salts on the solubility of vanillin [C4C1im][CF3SO3] 0.842 ± 0.024
at 303 K are depicted in Figure 5. The results for gallic acid are [C4C1im][N(CN)2] 1.335 ± 0.021* 1.013 ± 0.049 1.32
similar and are presented in the Supporting Information- Figure [C2C1im][N(CN)2] 0.819 ± 0.039
S4. These results emphasize the role of the ILs chemical [C4C1py][N(CN) 2] 3.721 ± 0.036# 1.238 ± 0.023 3.01
structure on their performance as hydrotropes, while [C4C1py]Cl 0.800 ± 0.058 0.775 ± 0.036 1.03
highlighting their superior capability when compared with
[C4C1pip]Cl 0.826 ± 0.037 0.716 ± 0.015 1.15
conventional hydrotropes and salting-in inducing salts, e.g.,
[C4C1pyrr]Cl 0.666 ± 0.023 0.660 ± 0.042 1.01
Na[C6H5O7], Na[C7H5O2], Na[TOS], Na[SCN] and Na[N(CN)2].
[N1112(OH)]Cl 0.323 ± 0.011
1.6 [N4444]Cl 1.321 ± 0.009 -0.731 ± 0.009 -1.81
[Vanillin] / mol.kg-1

#
[P4444]Cl 2.684 ± 0.007 -0.744 ± 0.007 -3.61
1.2 Na[SCN] 0.099 ± 0.002 -0.022 ± 0.001 -4.50
Na[C7H5O2] 0.515 ± 0.003 0.228 ± 0.003 2.26
0.8 Na[C6H5O7] -0.393 ± 0.008 0.559 ± 0.004 0.70
NaCl -0.126 ± 0.002 -0.078 ± 0.002 1.62
0.4 Na[TOS] 0.745 ± 0.034 0.427 ± 0.025 1.74
[N4444][TOS] 1.542 ± 0.065# 2.306 ± 0.096# 0.67
[P4441][TOS] 3.977 ± 0.098# 2.832 ± 0.035* 1.40
0.0
*Forms two liquid-liquid phases with IL aqueous solutions at concentrations above
0.0 0.4 0.8 1.2 1.6 10 wt %; # Forms two liquid-liquid phases with IL aqueous solutions at
[Hyd] / molHyd.kgwater-1 concentrations above 5 wt %.
Fig.5 Influence of the hydrotropes (ionic liquids and conventional salts)
concentration in the solubility of vanillin in aqueous solutions at 303 K: (---,)

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The higher the KHyd values, the higher is the ability of a given
compound to act as a hydrotrope. The hydrotropic capacity of
the different ILs and salts upon the solubility of vanillin follows
the order: Na[C6H5O7] > NaCl > Na[SCN] > Na[N(CN)2] >
[C2C1im]Cl > Na[C7H5O2] > [C4C1pyrr]Cl > [C4C1im]Cl > Na[TOS] >
[C4C1py]Cl > [C2C1im][N(CN)2] > [C4C1pip]Cl > [C6C1im]Cl >
[C4C1im][SCN] > [N4444]Cl > [C4C1im][N(CN)2] > [C8C1im]Cl >
[C10C1im]Cl > [C12C1im]Cl > [C14C1im]Cl > [N4444][TOS] >
[C4C1im][TOS] > [P4444]Cl > [C4C1py][N(CN)2 > [P4441][TOS]. This
pattern reflects the effects of the cation core, anion nature and
alkyl side chain length in ILs through their ability to act as
hydrotropes. Furthermore, the results obtained for gallic acid
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allow the evaluation of the effect of a broad range of cation
families and anions upon the hydrotropic effect. The hydrotropy
effect increases in the following order: [P 4444]Cl ≈ [N4444]Cl >>
NaCl > Na[SCN] > Na[C7H5O2] > [N1112(OH)]Cl > Na[TOS] >
Na[C6H5O7] > [C4C1im]Br > [C4C1pyrr]Cl > [C4C1im][SCN] >
[C4C1pip]Cl > [C4C1py]Cl > [C4C1im]Cl > [C4C1im][CH3SO4] >
[C4C1im][CF3SO3] > [C8C1im]Cl > [C4C1im][N(CN)2] >
[C4C1im][TOS] > [C4C1py][N(CN)2] > [N4444][TOS] > [P4441][TOS].A
detailed discussion on the effect of the chemical structures of
the ionic liquids cation and anion is presented below.
Effect of the ionic liquid cation
As depicted in Figure 6, the solubilities of vanillin and gallic acid
decrease only slightly in presence of NaCl, reflecting thus the
weak salting-out effect of this inorganic salt.
This trend suggests that neither Na+ nor Cl- have a significant
effect upon the solubility of the two antioxidants in water as Na +
and Cl- are both ranked in the middle of the Hofmeister series63.
Therefore, and in order to better evaluate the isolated impact
of given cations and anions, a series of sodium salts and chloride
ILs were used in this work.
The various cations studied cover aromatic (imidazolium and
pyridinium), cyclic non-aromatic (piperidinium and
pyrrolidinium), and non-cyclic non-aromatic (phosphonium and
ammonium). Figure 6 depicts the values of the hydrotropy
constants obtained for the solubility of vanillin and gallic acid in
aqueous solutions of Cl-based hydrotropes.With the exception
of the cholinium-based IL that seems to present only a small
effect upon the solubility of gallic acid (K Hyd=0.323 kg-1.mol), all
other ILs cations lead to an important hydrotropic effect. It
should be remarked that the quaternary ammonium- and
phosphonium-based ILs have opposite effects on the solubility
of vanillin and. gallic acid.
Although the formation of solute-hydrotrope complexes based
on ··· interactions has been used to explain the hydrotropy
concept14-17, the results here obtained clearly show that the
hydrotropic effect cannot be explained as resulting only from
··· interactions (between the aromatic antioxidant and the
aromatic cation cores) as the non-aromatic cations studied also
induce a significant increase on the antioxidants solubility.
Results that confirm this evidence were obtained, for instance,
with the non-aromatic [C4C1pip]Cl and [C4C1pyrr]Cl. This is in
good agreement with various authors8,13,64,65 that have
previously ruled out ··· interactions as the dominant effect
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governing the hydrotropy. This trend also discards the
View Article Online
possibility that the hydrotropy of ILs DOI: 10.1039/C5GC00712G
could be related to
clathrate formation as proposed by Rogers and co-workers66,67
Green Chemistry Accepted Manuscript
and Rebelo and co-workers68,69 to explain the enhanced
solubility of benzene in pure aromatic ILs. UV spectroscopy of
the solutions here studied also rules out the formation of
complexes as no changes in the spectra of the solute or the ILs
were observed (cf. the Supporting Information- Figure S5).
2.8
KHyd / kg.mol-1
2.2
1.6
1.0
0.4
-0.2
-0.8
Fig. 6 KHyd values for vanillin (red) and gallic acid (blue) using Cl-based hydrotropes.
It is also worth mentioning that the hydrotrope effect is often
solute specific, as shown by [N4444]Cl and [P4444]Cl, both
presenting large KHyd for vanillin but not for gallic acid. The
strong variations on the hydrotropy selectivities, calculated as
K (2)
Selectivity  HydVan K
HydGA
reported in Table 1, also clearly emphasize that the solute
specificity of the hydrotropes can be of high value to design
extraction and purification processes. In eq. 2, KHydVan and KHydGA
are the hydrotropy constants (kg-1.mol) of vanillin and gallic acid
respectively.
The results here reported reveal that, while most common
hydrotropes present aromatic anions in their constitution, the
hydrotropic effect can be induced by both aromatic and non-
aromatic cations. There are few cationic hydrotropes known 1,
and only in the past few years some sparse works using
quaternary ammonium-based salts were reported.70-72 In
summary, the results here obtained clearly support that ILs may
constitute a novel series of cationic hydrotropes.
Effect of the cation alkyl side chain length
Figure 7 depicts the variation on the solubility of vanillin in
aqueous solutions of [CnC1im]Cl ILs, with 2 ≤ n ≤ 14, along the
hydrotrope concentration.
The results depicted in Figure 7 clearly show two different
behaviors. For ILs from [C2C1im]Cl to [C6C1im]Cl, the hydrotropy
constant increases with the cation alkyl side chain length, with
the solubility following an exponential dependency with the
hydrotrope concentration, as predicted by the Setchesnow
equation. On the other hand, for the ILs with longer alkyl side
chains ([CnC1mim]Cl, n = 8 – 14) the solubility of vanillin along
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with the IL concentration no longer obeys Eq. (1) and all the other hand, sodium chloride and sodium thiocyanate have
View Article no
Online
compounds have a similar impact upon the solubility significant effect upon the solubility ofDOI:
the10.1039/C5GC00712G
two antioxidants
irrespective of their alkyl side chain lengths. These two patterns even with the latter inducing a slight salting-in behaviour.

Green Chemistry Accepted Manuscript

suggest that two different mechanisms of enhanced solubility
are at play on these systems. For the lighter compounds 1.8

KHyd / kg.mol-1
([CnC1mim]Cl, n = 2 – 6) the hydrotropic behavior is witnessed.
As it results from the formation of co-aggregates between the
solute and the hydrotropes, as discussed below in more detail, 1.2
the hydrotropic efficiency increases with the cation alkyl chain
length.2,56,73
0.6
However, for ILs with self-aggregation ability in aqueous
solution (above [C8C1mim]Cl74), the hydrotropy is replaced by a
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micellar solubilization mechanism. For these compounds, the 0.0

enhancement on solubility is very active at low concentrations
of IL, close to the critical micellar concentration, but quickly
seems to saturate at around 0.2 mol.kg -1, above which a much -0.6
weaker effect upon the solubility is observed. Indeed, for higher
hydrotrope concentrations (around 1-1.2 molIL.kgwater-1), the
hydrotropic effect of [C6C1mim]Cl upon the solubility of vanillin
becomes more important than that observed with ILs of longer
alkyl side chains.
1.4
log (S/S0)

1.2 Fig. 8 KHyd values for vanillin (red) and gallic acid (blue) using [C 4C1im]- and Na-
based hydrotropes.
1.0
0.8 Most conventional hydrotropes have ammonium, calcium,
0.6 potassium or sodium as counter ions,4 but the effect of the
0.4 counter ion is seldom explored. However, the results here
reported reveal that the role of the counterion could be highly
0.2
relevant, as demonstrated by the KHyd values presented in Table
0.0
1.
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
[Hyd] / molHyd.kgwater For vanillin, the hydrotropic efficiency of the anion of the
-1
[C4C1im]-based ILs follows the series:
Fig. 7 Influence of the ionic liquids concentration in the vanillin solubility at 303 K:
(), pure water, () [C2C1im]Cl, () [C4C1im]Cl, () [C6C1im]Cl, () [C8C1im]Cl, Cl- < [SCN] - < [N(CN)2]- < [TOS]-
() [C10C1im]Cl, () [C12C1im]Cl, and (▬) [C14C1im]Cl.
while for gallic acid, the trend on the enhanced solubility by the
These results are in good agreement with the results by Shimizu [C4C1im]-based ILs follows the rank:
and collaborators23-24, that based on the Kirkwood-Buff (KB) Br-< [SCN]- < Cl-< [CH3SO4]- < [CF3SO3]-< [N(CN)2]- < [TOS]-.
theory of solutions, shown that “micelle formation reduces the Knowing that NaCl induces a minor salting-out of gallic acid and
solubilization efficiency per hydrotrope molecule”. vanillin in aqueous media, the anions in compounds displaying
a KHyd lower than chloride have thus a deleterious effect upon
Effect of the ionic liquid anion the cation hydrotropicity. Chloride and all the anions to its left
on these series do not present a hydrotropic behaviour and thus
Most hydrotropes industrially used are anionic, and often do not contribute to enhance the solubility of the studied
contain a phenyl group.4 Along with the evaluation of ILs as biomolecules. Dycianamide and tosylate anions display,
hydrotropes, the effect of some common hydrotropes was also however, the opposite effect. Tosylate, a common name for p-
ascertained. toluene sulfonate, is indeed a well-known hydrotrope1-4; yet,
According to the results presented in Table 1 and Figure 8, the the action of [N(CN) ]- as hydrotrope is, to the best of our
2
only salts that seem to display hydrotropic activity for both knowledge, here reported for the first time. Both anions
biomolecules are sodium benzoate (KHyd of 0.514 kg-1.mol for contribute to a further enhancement of the cation
vanillin and 0.228 kg-1.mol for gallic acid) and sodium tosylate hydrotropicity in a synergistically, almost additive, manner.
(KHyd of 0.746 kg-1.mol for vanillin and 0.428 kg -1.mol for gallic While in the previous section it was shown that ILs are a novel
acid), i.e., those with an aromatic anion. The sodium citrate is class of cationic hydrotropes, these results suggest that when
only able to enhance the solubility of gallic acid (KHyd = 0.560 kg- combined with an anion with hydrotropic capacity an even
1.mol) whereas it seems to decrease the solubility of vanillin
more powerful catanionic hydrotrope is generated.
(KHyd = -0.393 kg-1.mol) in aqueous solutions. As shown in Figures 3 and 8, the hydrotropic effect of the
These results again confirm that the hydrotropicity may be studied compounds is, in general, stronger upon the solubility
strongly solute dependent, allowing thus the design of an of vanillin than of gallic acid. The K of vanillin is 1.2275 while
ow
extraction process based on the hydrotrope selectivity. On the

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for gallic acid it is 0.72,75 suggesting that the hydrotropic
solubilisation may be more effective for more hydrophobic
solutes. Yet, this general rule may be overruled by specific
interactions that could explain the significant differences in
solubility observed between the two compounds, suggesting
that the hydrotrope-solute interactions on the gallic acid
aggregates are of a different nature than those with vanillin,
being this the cause for the hydrotrope selective enhancement
of solubility. The results reported in Table 1 suggest also that
the cation and anion may have different roles in the hydrotropy,
with the cation effect being dominated by the hydrophobicity
of the solute while the anion seems to present specific
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interactions. This may however be related to the specific nature
of the solutes here studied and their hydrogen bond donor
abilities that can be matched by the anions hydrogen bond
acceptor nature that is absent in the cations.
In summary, the results here reported clearly show that the
capability of ILs to act as hydrotropes selectively enhancing the
solubility of each compound depending on the IL
characteristics, on the solute hydrophobicity and, in general, on
how the solute interacts with and participates in the formation
of aggregates in aqueous solutions of ILs as discussed below.
A curious and unexpected outcome was observed for some of
these mixtures for which, above a critical concentration of IL, a
biphasic system was formed as shown in Figure S6 (cf. the
Supporting Information). The systems, for which two phases
were observed, are reported in Table 1. As the solute does not
present a salting-out inducing nature, which could explain the
biphasic system formation under the same light of the
formation of IL-based aqueous biphasic systems (ABS)30, the
coexisting phases were quantified by UV-spectroscopy and
NMR spectroscopy was employed in order to understand the
phase separation and shed further light upon the mechanism of
hydrotropicity evidenced in this work.
The top and bottom phases of the biphasic system created by
vanillin, [C4C1im][N(CN)2] and water were quantified by UV-
spectroscopy, and the bottom phase revealed to have twice the
concentration of IL and 5 times more vanillin than the top
phase. Aqueous solutions of [N4444][TOS] and [P4441][TOS], at
concentrations approximately of 0.28 mol IL.kgwater-1 and 0.62
molIL.kgwater-1, respectively, were also tested for the
solubilisation of vanillin and gallic acid. In all situations, the
system separated into two liquid phases. For the first system,
the bottom phase showed almost 21 times more [N 4444][TOS]
and 18 times more vanillin than the top phase. For the second
system, the bottom phase presented almost 20 times more
[N4444][TOS] and 12 times more vanillin than the top phase. To
gather further insights into the molecular-level mechanisms
responsible for the phase separation at given concentrations of
hydrotrope and antioxidant, NMR spectra were collected for
each phase. All the systems analysed further confirm that,
unlike for IL-based ABS, there is not a significant separation of
the IL and demixing inducer between the two aqueous phases.
These observations suggest that, unlike the pure IL that is
hydrophilic and thus completely water miscible, the IL+solute
aggregates, whose presence is demonstrated and discussed
below, are of a different nature, being more hydrophobic and
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not fully water miscible, leading therefore to the View
formation of
Article Online
two liquid phases: one water-rich phaseDOI: 10.1039/C5GC00712G
with little solute and
hydrotrope (IL) and another phase rich in hydrated IL where the
Green Chemistry Accepted Manuscript
biomolecules are highly soluble.
Effect of temperature
The temperature impact on the hydrotropic solubility of the
biomolecules investigated was also evaluated. For that purpose,
the solubility of vanillin was investigated in aqueous solutions
of [C2C1im][N(CN)2] and sodium benzoate. Figure 9 shows the
effect of temperature upon the KHyd values between 303 and
323 K.
1.2
KHyd / kg.mol-1
0.9
0.6
0.3
0.0
300 305 310 315 320 325
T/K
Fig. 9 KHyd temperature dependency for the solubility of vanillin in aqueous
solutions of () [C2C1im][N(CN)2] and () [Na][C7H5O2].
The data concerning the influence of [C2C1im][N(CN)2] and
sodium benzoate on the solubility of vanillin at 303, 313 and 323
K are presented in Supporting Information- Table S3.
In general, a significant increase in the solubility of vanillin with
an increase in temperature is observed. This enhanced solubility
promoted by hydrotropes is even more striking when compared
with the increase of the solubility of vanillin in water with
temperature, which is quite low as shown in the Supporting
Information- Figure S7. This trend, resulting from a decrease in
size and increase in number of the aggregates with temperature
as revealed by DLS, suggests that the effects of the hydrotrope
concentration and temperature can be combined to achieve a
maximum effect at both the solubilisation and the recovery of
the solute, as discussed below.
Dynamic Light Scattering.
In order to probe the mechanism of hydrotropy previously
highlighted, some of the aqueous solutions containing vanillin
and hydrotropes we analysed by dynamic light scattering (DLS).
This study started by the evaluation of the presence of
aggregates in aqueous solutions of IL or vanillin. No aggregates
were observed in aqueous solutions of [C 4C1im][TOS] at 0.64
molIL.kgwater-1 or vanillin close to saturation (after
homogeneization of the mixture on an ultrasonic bath at room
temperature). DLS measurements were then performed for
solutions of variable concentrations of vanillin in 0.64
molIL.kgwater-1 of [C4C1im][TOS]. The results depicted in Figure 10
confirm the presence of aggregates that increase in size with an
increase of the vanillin concentration.
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An aqueous solution of vanillin at 0.73 mol.kgwater-1 and IL at 0.80
mol.kgwater-1 was then diluted with pure water aiming at
evaluating the change in the size of aggregates at a fixed
vanillin/IL ratio. As shown in Figure 10, an increase in the size of
aggregates with the vanillin/IL concentration is also observed.
These results clearly establish the presence of aggregates in
aqueous solution, but only when both the hydrotrope and
vanillin are present, and show that the molecular mechanism
which dominates the hydrotropy is related with the formation
of hydrotrope-solute aggregates. Dhinakaran et al.42 proposed
that the increased solubility of vanillin in conventional
hydrotrope solutions is justified as a collective molecular
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phenomenon, possibly occurring by the aggregation of the
solute with the hydrotrope due to improved hydrogen-bonding.
The most detailed and comprehensive studies on the
hydrotropic solubilisation were recently reported by Shimizu
and co-workers.10,23,24 They clearly established, based on the
interpretation of experimental data using the fluctuation theory
of solutions, that the mechanism of hydrotropic solubilisation is
dominated by the hydrotrope-solute aggregation.23 In the same
line, the results here reported show that the solute molecules
must take part in the aggregation process of the hydrotrope,
thus forming co-aggregates with the hydrotrope molecules in
aqueous solution. The formation of a stable co-aggregate
depends on the molecular structure of both cation and anion,
as well as on the functional group(s) of the solute, that would
control their interactions further responsible for the formation
of stable aggregates. The variation of the aggregate size when
the ratio vanillin/IL is changed (yellow circles in Figure 10), or
kept constant (green triangles in Figure 10) suggests however
that the aggregates formation does not result from a
stoichiometric complex between vanillin and the IL, unlike was
previously observed by some authors for the solubility of
benzene in aromatic ILs.66-69
5.0
Peak / nm
4.0
3.0
2.0
1.0
0.0
0.2 0.4 0.6
[Vanillin] / mol.kg-1
Fig. 10 Aggregates size change with the vanillin concentration: () constant
hydrotrope concentration, i.e., different vanillin/IL ratios, and () constant ratio
between the vanillin and hydrotrope concentrations.
Nuclear Magnetic Resonance
1H-NMR
spectra were collected for aqueous solutions of
[C4mim]Cl in a concentration range from 0.1 up to 5.73 mol.kg -
1 (pure IL). The results are presented in Figure 11 and show
regular chemical shift deviations with the protons of the ring
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region presenting negative deviations due to the favorable
View Article Online
DOI: 10.1039/C5GC00712G
polar interactions with water, in particular the C(2) proton
which presents a very large shift resulting from its hydrogen
Green Chemistry Accepted Manuscript
bond with the solvent. The protons of the alkyl chain, in
particular those of C(7), C(8) and C(9) have the opposite type of
deviations as they have poor interactions with water as
expected.
The 1H NMR spectra of ternary mixtures water-[C4C1im]Cl-
vanillin were collected for mixtures at fixed vanillin
concentrations of 0.05 mol·kg-1 and 0.13 mol·kg-1 while varying
the IL concentration from 0.1 to 1.4 mol·kg -1. As shown in
Supporting Information- Figure S8, no significant chemical shift
deviations of the vanillin were observed in any of these
solutions. 1H NMR spectra were also collected for vanillin
solutions with concentrations up to 0.43 mol·kg -1 prepared from
an initial aqueous solution of IL at 1.13 mol·kg -1. The chemical
shift deviations of the IL protons are presented also in Figure 11
within the blue ellipse. Unlike what was observed for the vanillin
that seems to suffer no effect from the IL in a much broader
concentration region, the effect of the vanillin upon the IL is
quite significant.
0.8
0.4
ΔδH / ppm
0.0
-0.4
5
-0.8 4 8
9
N+
-1.2
10
Cl-Br
-
N
3 7
2 6
-1.6
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0
[IL] / mol.kg-1
Fig. 11 1H NMR chemical shift deviations of [C 4C1im]Cl in aqueous solutions relative
to pure [C4C1im]Cl (5.7 mol·kg-1 ): () H(2), () H(4), () H(5), () H(6), () H(7),
() H(8), () H(9), and () H10. Effect of the vanillin concentration within the
blue ellipse.
While the protons on the polar region further increase their
deviations from what was observed due to the mixture with
water, suggesting that novel polar interactions between the
vanillin and the ring protons are happening, and as will be
confirmed below by the MD simulations, the chemical shifts of
the protons on the alkyl chain have the opposite behaviour,
with their deviations decreasing and approaching the values for
the pure IL, suggesting thus that the environment of the alkyl
chains becomes again less polar. These results suggest that the
chains may be interacting with the non polar regions of vanillin
0.8 or organizing themselves into non-polar domains. The
molecular picture that can derive from the 1H NMR spectra is
one of the reorganization of the IL cations in solution due to the
presence, and their interactions with, vanillin molecules.
Molecular Dynamics Simulations
The existence of co-aggregates between the hydrophobic solute
and the IL ions in aqueous solution was further probed by
Molecular Dynamics simulations. Three types of model system
were considered: i) aqueous solutions containing the
hydrotrope (ILs based on the 3-butyl-1-methylimidazolium
cation combined with either the thiocyanate or the
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dicyanamide anions, [C4C1im][SCN] or [C4C1im][N(CN]2]); ii) View Article Online

aqueous solutions containing the vanillin hydrophobic solute; DOI: 10.1039/C5GC00712G
and iii) aqueous solutions containing both the hydrotrope and

Green Chemistry Accepted Manuscript

the hydrophobic solute species. Simulation boxes containing
the pure IL were also produced in order to act as a reference for
the aggregation patterns of the ionic species. All molecules/ions
were modelled according to well-known and tested atomistic
force-fields commonly used for mixtures of ionic liquids with
molecular solutes/solvents (cf. Experimental Section).
In order to match the concentration ranges of the aqueous
solutions tested experimentally, we used simulation boxes i)
containing 101 [C4C1im][N(CN]2] or 105 [C4C1im][SCN] ion pairs
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together with 4600 water molecules, corresponding to identical

IL weight fractions of 0.2000, or to concentrations of 1.220 and
1.268 mol.kg-1, respectively; ii) containing 100 vanillin
molecules in 4600 water molecules (this is well above the
solubility limit of vanillin in pure water and, as expected, the
simulations yielded a vanillin-rich droplet segregated from a
water-rich phase); and iii) 100 vanillin molecules combined with
101 or 105 hydrotrope ion pairs and 4600 water molecules.
Taking into account the KHyd values listed in Table 1 for the
[C4C1im][N(CN]2] and [C4C1im][SCN] hydrotropes (0.978 and
1.335 kg-1.mol), the simulated vanillin-IL-water mixtures should
contain a single phase in the case of the [C 4C1im][N(CN]2]
hydrotrope mixture and two phases in the case of the
[C4C1im][SCN] hydrotrope mixture. The use of relatively large
concentrations of hydrotrope and vanillin in the simulation
boxes (close or above the solubility limit of vanillin in the Fig. 12 Simulation snapshots and discrete probability distribution functions of
aggregate sizes, P(na) for different types of system and aggregate type. (a-b) Ionic
aqueous solutions) is related to the necessity of having liquid aqueous solutions; (c) Water-vanillin mixture; (d-e) Vanillin in ionic liquid
aqueous solutions. (green graphs): ionic liquid polar aggregates (strands); (blue
adequate statistics in the MD run trajectories and ensuing graphs): anion-water network; (red graphs) vanillin clusters.

structural and aggregate analyses. Moreover, by comparing the

different systems at similar IL and vanillin mole or weight
fractions allowed us to compare directly the distinctive
aggregation patterns that occurred in the different situations.
Figure 12 shows five snapshots of the equilibrated simulation
boxes. Boxes a and b highlight the structure of diluted solutions
of the hydrophilic [C4C1im][N(CN]2] and [C4C1im][SCN] ILs in
water: the continuous polar network that is the hallmark of
most pure ionic liquids is broken into smaller aggregates, that
nevertheless can still incorporate in some cases a few tens of
alternating ions —the size distribution probability functions,
P(na), of those polar aggregates can be seen in the graphs with
green bars in Figures 12a and 12b.
In other words, the “dissolved and diluted” IL is not simply a set
of isolated ions solvated by water molecules, but rather a series
of small (and not so small) filamentous ionic strands
incorporated within the liquid water matrix. Aggregate analyses
of those strands show that the number of neighbours of each
ion in a given strand (calculated using the Ni(na) described in the
Experimental section) is never much larger than two, which
confirms the non-branched and filamentous nature of the
aggregates. Moreover, their estimated volume-to-length ratio,
RV(na)/Rd(na), is generally below 0.03, which means that their
shapes can be approximated to elongated prolate ellipsoids.
The incorporation of such strands in the aqueous solution can
be further probed by the analysis of anion-water aggregates
(graphs with blue bars in Figures 12a and 12b) that show that
the interactive centers of the anions (the nitrogen atoms of
dicyanamide and the sulphur and nitrogen atoms of
thiocyanate) undergo specific H-bond-type interactions with

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the hydrogen atoms of the water molecules and help to create
small networks of alternating water molecules and anions that
can contain a few tens of species. It is interesting to notice that
whereas around 80% of the water molecules do not contact any
anion (at the tested concentrations the ionic liquid only
occupies less than 20% of the total volume of the solution), the
remainder 20% of the water molecules become part of clusters
that are composed of several anions. In fact, the size
distribution of the clusters mimics the distribution of the IL
clusters present in the solution. In other words, the interactions
between the anions and the water molecules help to stabilize
those filamentous IL clusters in the midst of the aqueous
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solution. This complex and unique structure will play an
important role in terms of hydrotropy (see below).
It must also be stressed that, unlike the polar moieties of the
ions, the alkyl side chains of the cations (butyl groups in the
present case) do not form noticeable aggregates. The
corresponding aggregate size distribution functions (not shown)
indicate that around 65% of the butyl chains remain isolated
from each other, some 24% form pairs, and the remainder 11%
are aggregated into clusters with just 3-5 chains. This confirms
that the role of these short-tailed ILs as hydrotropes cannot be
attributed to surfactant-type effects such as the possibility of
micelle formation.
Figure 12c shows the above-mentioned vanillin-rich droplet
segregated from a water-rich phase. In this case, the MD
trajectory evolved naturally to a situation of liquid-liquid
demixing (albeit at a nanometric scale due to the size limitations
of the simulated system) and illustrates nicely the difference
between the one-phase situation of the hydrophilic IL aqueous
solutions (boxes a and b) and the two-phase split that occurs in
the hydrophobic solute system (box c). It is important to notice
that both types of system have similar solute concentrations.
The graph in figure 12c shows the size distribution function of
aggregates containing vanillin molecules. The distribution
shows that most vanillin molecules can be found in very large
aggregates comprising most of the vanillin molecules present in
the system (one of such droplets is the one represented in the
snapshot) in equilibrium with a few vanillin molecules isolated
in the aqueous phase (the small bars in the left-hand side of the
graph). This distribution illustrates the aforementioned phase
separation. The average number of contact neighbours, Ni, of a
given vanillin molecule within the droplet is around 9.8.
Figures 12d and 12e show hydrotrope effects in action, when an
aqueous solution containing an ionic liquid is mixed with
vanillin. Both snapshots illustrate that the integrity of original
vanillin droplet (Figure 12c) is broken and several fragments are
solvated by the hydrotrope-water solution. Interestingly, the
distribution functions of the polar aggregates and the anion-
water network (green and blue graphs, respectively) show only
small shifts to smaller aggregation sizes relative to the
analogous functions of Figures 12a and 12b. This means that the
small ILs strands stabilised by their water-anion interactions
continue to exist in the presence of the vanillin molecules. On
the other hand, the vanillin aggregate distribution functions
(red graphs) show noticeable shifts relative to the situation in
Figure 12c: the presence of the hydrotrope is promoting the
12 | J. Name., 2012, 00, 1-3
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Journal Name
mixing of the vanillin molecules in the aqueousView phase. The
Article Online
average number of vanillin neighbours, DOI: 10.1039/C5GC00712G
Ni, within the broken
vanillin clusters drops from the original 9.8 value to average
Green Chemistry Accepted Manuscript
values of 5.3 and 5.2 in the aqueous [C4C1im][N(CN]2] and
[C4C1im][SCN] systems, respectively: the clusters are not only
smaller but are also intermeshed with other species.
Fig. 13 Discrete probability distribution functions of aggregate sizes, P(na), and (as
insets) pair radial distribution functions, g(r), for different types of aggregate type
and pairs of interaction centre. (a-b) size distribution of cation-vanillin aggregates
and cation-vanillin interactions in the systems with (a) [N(CN)2]-induced and (b)
[SCN]-induced hydrotropy. (c-d) size distribution of anion-vanillin aggregates and
anion-vanillin interactions in the systems with (c) [N(CN) 2]-induced and (d) ) [SCN]-
induced hydrotropy.
In order to check the origin of the hydrotrope effect one must
analyse in detail the interactions between the IL strands and the
vanillin molecules.
Figure 13 shows relevant aggregate distribution functions along
with selected radial distribution functions that probe the
intensity of different ion-vanillin interactions. Figures 13a and
13b show the aggregate size distribution functions
corresponding to the alternate clustering of 1-butyl-3-
methylimidazolium cations and vanillin molecules.
The “van-cat” distribution function of figure 13a corresponding
to the [C4C1im][N(CN]2] aqueous solution shows that only 11%
of the cations or vanillin molecules are not in contact with the
other species and that most of them are part of very large
clusters with 140 < na < 185 (with a maximum probability
around na = 170).
These mixed cation-vanillin clusters are formed due to
favourable interactions between the two species, not only
dispersion forces between their less polar moieties (the butyl
chain of the cation, the aromatic ring and ether group of
vanillin), but also between specific interaction centres located
in each of them. One such interaction —between the oxygen
atom of the hydroxyl group of vanillin (OH) and the most acidic
aromatic hydrogen atom of the imidazolium ring (HCR)— is
quantified in the inset of Figure 13a that shows the
corresponding pair radial distribution function. Integration
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below the first peak of the g(r) function shows that about 35%
of the vanillin-cation contacts involve a OH-HCR interaction.
Figure 13b shows that the vanillin-cation clusters in the
[C4C1im][SCN] aqueous solutions (120 < na < 185, with a
maximum probability around na = 160) are not as big as those
found in the [C4C1im][N(CN]2] solutions. The comparison of the
two insets of Figures 13a and 13b also point out to slightly
weaker OH-HCR interactions in the latter case. This is consistent
with the lower KHyd value found for the [C4C1im][SCN] solutions,
when compared with that of the [C 4C1im][N(CN]2] solutions.
The difference between the two hydrotropes is even more
noticeable if one compares the vanillin-anion cluster
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distributions and the corresponding g(r) functions (Figures 13c
and 13d). In both situations, the vanillin-anion cluster sizes are
always smaller than the corresponding vanillin-cation clusters.
This is due to the fact that the anion is also involved in strong
interactions with the water solvent and is responsible for the
stabilization of the IL strands in the midst of the aqueous
solution. Nevertheless, the dicyanamide anion is more able to
form larger clusters with the vanillin molecules than its
thiocyanate counterpart. Those larger clusters are a
consequence of the stronger H-bond-type interactions involving
the hydrogen atom of the hydroxyl group of vanillin and the
nitrogen atoms of the [N(CN]2] anion (inset of Figure 13c),
relative to the same type of interactions involving the nitrogen
and sulphur atoms of the [SCN] anion (inset of Figure 13d).
In summary, the present MD simulations can explain, from a
molecular perspective, how short-chained hydrophilic ionic
liquids can act as hydrotropic agents: i) in the concentration
range where hydrotrope effects are noticeable, hydrophilic ILs
are not completely dissociated but form ionic aggregates
(strands) of a few tens of ions; ii) those strands are stabilized in
the aqueous medium by anion-water interactions; iii) most of IL
cations are organic in nature and can therefore interact with
organic (hydrophobic) solutes. Such interactions are not
restricted to dispersion forces between the nonpolar moieties
of the cation and the solute molecules (which can nevertheless
be quite important) but can also involve other specific
interactions (e.g. between H-donors and H-acceptors and 
interactions); iv) the formation of cation-solute aggregates will
promote the dissolution of the hydrophobic solute in the midst
of the ionic liquid strands; v) since these strands are anchored
to the aqueous solution via their anion-water interactions, the
end result will be the dissolution/dispersion and stabilization of
the organic solute molecules in the aqueous solution.
Recovery of solutes from solution
The results of enhanced solubility in aqueous solutions of ILs
here reported also suggest that the high performance
demonstrated by these aqueous solutions in the extraction of
biocompounds from biomass31 may be a major outcome of this
enhanced solubility created by the hydrotropic behaviour of the
ILs, and not only from the biomass disruption as commonly
accepted.76,77 This being the case, the nature of the hydrotropic
solutions discussed above can thus be used not only for the
extraction but also for the recovery of the target compounds
This journal is © The Royal Society of Chemistry 20xx
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COMMUNICATION
from aqueous media. Since the solubility of theView antioxidants
Article Online
(and any other solutes) largely depends DOI: 10.1039/C5GC00712G
on the IL (hydrotrope)
concentration, and their solubility is far superior to their
Green Chemistry Accepted Manuscript
saturation values in pure water, the results obtained combined
with the hydrotropy concept suggest that the recovery of the
solutes from the hydrotrope aqueous solution can be attained
by a simple dilution with water, combined or not with a
decrease of temperature.
Pure vanillin a)
Vanillin after precipitation d)
a) b) c) d)
Fig. 14. a) appearance of commercial vanillin and respective 1H NMR spectrum; b)
vanillin dissolved in 0.80 molIL.kgwater-1aqueous solution of [C 4C1im][TOS] at 303 K;
c) precipitation of vanillin with the addition of water and decrease of temperature;
and d) recovered vanillin and respective 1H NMR spectrum.
Since the vanillin solubility is extremely sensitive to the
hydrotrope concentration and temperature, as shown before,
some tests on the recovery of vanillin from aqueous solution by
further dilution with water were carried out. For this purpose,
an aqueous solution with 0.81 mol.kgwater-1 of [C4C1im][TOS] was
prepared and saturated with vanillin (0.82 mol.kg-1 = 125 g.kg-1)
at 303 K.
To recover the vanillin, an equal volume of water was added to
the vanillin-IL-water solution aiming at decreasing the
hydrotrope concentration to half of its initial value. The
decrease of the hydrotrope concentration in solution from 0.81
to 0.36 mol.kgwater-1 led to the precipitation of (59 ± 1) wt% of
the initial vanillin in solution (Figure 14).
The expected value for the vanillin recovery, calculated from
the solubility curves previously measured, is 61%, which is in
good agreement with the experimental results obtained.
The precipitated vanillin was recovered by filtration, washed
with cold water and dried. The macroscopic appearance of the
recovered vanillin is the same as the initial compound, while its
purity was ascertained and confirmed by 1H NMR (Figure 14).
These simple essays show how the compounds dissolved by
hydrotropic action can be recovered using water as anti-solvent
to induce their precipitation. This behaviour can be used with
remarkable advantages on the field of IL-mediated extractions
of added-value compounds from biomass. In fact, it is well-
known that IL aqueous solutions are excellent solvents for the
extraction of alkaloids, triterpenoids, flavonoids, among others,
from natural matrices.31 Although several works demonstrated
the high potential of IL aqueous solutions, the
isolation/purification of the target compounds as well as the
recovery of the solvents for further use has been seldom
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studied.31 The approach proposed here - the use of a green
solvent like water as anti-solvent – certainly will allow the
design of novel strategies for the recovery of both the added-
values compounds and IL solvents.
Conclusions
This comprehensive study regarding the solubility of
biomolecules in aqueous solutions of ILs disclosed, for the first
time, the ability of ILs to act as hydrotropes. It is here clearly
established that ILs are a novel class of catanionic hydrotropes
with a superior performance since both the IL cation and anion
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may contribute to enhance the solubility of poorly soluble
compounds in aqueous solution. The overall results using DLS,
NMR and MD support the idea that the enhanced solubility of
sparingly soluble solutes in water is driven by the formation of
solute-IL aggregates.
The results here reported are also essential to the
understanding of the mechanisms which rule the high-
performance extraction of biomolecules from biomass using IL
aqueous solutions. It shows that contrarily to what was
proposed by us76 and others,77 the high efficiency achieved
using IL aqueous solutions on the extraction of added-value
compounds from biomass31 is not only related with the
disruption of the biomass structure, but also with the enhanced
solubility of the target compounds in aqueous IL solutions, i.e.,
with the hydrotropic role displayed by ILs in aqueous media.
Given the selective character of the hydrotropic effect, also
revealed in this work, the use of hydrotropes further allows a
selective extraction of the target compound. When using
hydrotropes in the extraction of biocompounds it is possible to
develop a simple and benign biomolecule recovery process,
simple by using water as the anti-solvent to induce the
precipitation/recovery of the extracted molecules and IL
solvents.
Acknowledgements
This work was developed in the scope of the projects CICECO-
Aveiro Institute of Materials (Ref. FCT UID/CTM/50011/2013)
and EXPL/QEQ-PRS/0224/2013 financed by national funds
through the FCT/MEC and when applicable co-financed by
FEDER under the PT2020 Partnership Agreement. A.F.M.
Cláudio acknowledges FCT for the PhD grant
SFRH/BD/74503/2010. M. G. Freire acknowledges the European
Research Council (ERC) for the Starting Grant ERC-2013-StG-
337753.
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DOI: 10.1039/C5GC00712G
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Green Chemistry Accepted Manuscript

Ionic liquids are powerful catanionic hydrotropes with cation and anion synergistically contributing to increase the solubility of
biomolecules in water.